Two-photon directed evolution of green fluorescent proteins (original) (raw)
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The journal of physical chemistry letters, 2017
Fluorescent proteins (FPs) are indispensable markers for two-photon imaging of live tissue, especially in the brains of small model organisms. The quantity of physiologically relevant data collected, however, is limited by heat-induced damage of the tissue due to the high intensities of the excitation laser. We seek to minimize this damage by developing FPs with improved brightness. Among FPs with the same chromophore structure, the spectral properties can vary widely due to differences in the local protein environment. Using a physical model that describes the spectra of FPs containing the anionic green FP (GFP) chromophore, we predict that those that are blue-shifted in one-photon absorption will have stronger peak two-photon absorption cross sections. Following this prediction, we present 12 blue-shifted GFP homologues and demonstrate that they are up to 2.5 times brighter than the commonly used enhanced GFP (EGFP).
Two-photon absorption properties of fluorescent proteins
Nature Methods, 2011
Two-photon excitation of fluorescent proteins is an attractive approach for imaging living systems. Today researchers are eager to know which proteins are the brightest and what the best excitation wavelengths are. Here we review the two-photon absorption properties of a wide variety of fluorescent proteins, including new far-red variants, to produce a comprehensive guide to choosing the right fluorescent protein and excitation wavelength for two-photon applications.
The Journal of Physical Chemistry B, 2009
Fluorescent proteins with long emission wavelengths are particularly attractive for deep tissue twophoton microscopy. Surprisingly little is known about their two-photon absorption (2PA) properties. We present absolute 2PA spectra of a number of orange and red fluorescent proteins, including DsRed2, mRFP, TagRFP, and several mFruit proteins, in a wide range of excitation wavelengths (640-1400 nm). To evaluate 2PA cross section (σ 2 ), we use a new method relying only on the optical properties of the intact mature chromophore. In the tuning range of a mode-locked Ti:sapphire laser, 700-1000 nm, TagRFP possesses the highest two-photon cross section, σ 2 = 315 GM, and brightness, σ 2 φ = 130 GM, where φ is the fluorescence quantum yield. At longer wavelengths, 1000-1100 nm, tdTomato has the largest values, σ 2 = 216 GM and σ 2 φ = 120 GM, per protein chain. Compared to the benchmark EGFP, these proteins present 3-4 times improvement in two-photon brightness.
ChemPhysChem, 2005
E 2 GFP = green fluorescent protein mutant We report the two-photon excitation and emission of a recently developed green fluorescent protein (GFP) mutant, E 2 GFP. Two main excitation bands are found at 780 and 870 nm. Blinking and irreversible and reversible bleaching were observed. Fluorescence blinking occurs in the millisecond range and has been ascribed to conversions between the neutral, anionic and dark zwitterionic states. Bleaching is observed after approximately 10 to 4000 ms depending on the excitation power, and it is probably due to a conversion to a dark state. The striking feature of this GFP mutant is that the fluorescence can be recovered with very high efficiency only upon irradiation at 720 AE 10 nm. This GFP mutant therefore seems promising as an almost permanent chromophore for two-photon excitation (TPE) microscopy or for applications in single-molecule memory arrays.
A new approach to dual-color two-photon microscopy with fluorescent proteins
BMC Biotechnology, 2010
Background: Two-photon dual-color imaging of tissues and cells labeled with fluorescent proteins (FPs) is challenging because most two-photon microscopes only provide one laser excitation wavelength at a time. At present, methods for two-photon dual-color imaging are limited due to the requirement of large differences in Stokes shifts between the FPs used and their low two-photon absorption (2PA) efficiency.
Visible-wavelength two-photon excitation microscopy for fluorescent protein imaging
Journal of Biomedical Optics, 2015
The simultaneous observation of multiple fluorescent proteins (FPs) by optical microscopy is revealing mechanisms by which proteins and organelles control a variety of cellular functions. Here we show the use of visible-light based two-photon excitation for simultaneously imaging multiple FPs. We demonstrated that multiple fluorescent targets can be concurrently excited by the absorption of two photons from the visible wavelength range and can be applied in multicolor fluorescence imaging. The technique also allows simultaneous single-photon excitation to offer simultaneous excitation of FPs across the entire range of visible wavelengths from a single excitation source. The calculation of point spread functions shows that the visible-wavelength two-photon excitation provides the fundamental improvement of spatial resolution compared to conventional confocal microscopy.
One-Photon and Two-Photon Excitation of Fluorescent Proteins
Springer Series on Fluorescence, 2011
Fluorescent Proteins (FPs) offer a wide palette of colors for imaging applications. One purpose of this chapter is to review the variety of FP spectral properties, with a focus on their structural basis. Fluorescence in FPs originates from the autocatalytically-formed chromophore. Several studies exist on synthetic chromophore analogs in gas phase and in solution. Together with the X-ray structures of many Fluorescent Proteins, these studies help to understand how excitation and emission energies are tuned by chromophore structure, protonation state, and interactions with the surrounding environment, either solvent molecules or aminoacids residues. The increasing use of Fluorescent Proteins in two-photon microscopy also prompted detailed investigations of their two-photon excitation properties. The comparison with one-photon excitation reveals non-trivial features, which are relevant both for their implications in understanding multiphoton properties of fluorophores and for application purposes.
Searching for the two-photon brightest red fluorescent protein and its optimum excitation wavelength
Society of Photo Optical Instrumentation Engineers Conference Series, 2009
We study 2PA spectra of red fluorescent proteins (FPs), including DsRed2, mRFP, tagRFP, and mFruits in a wide range of excitation wavelengths, 600 - 1200 nm. For evaluation of mature FP extinction coefficient and concentration we propose a pure optical method which is based on Strickler - Berg equation, relating fluorescence radiative lifetime with molecular extinction coefficient. 2PA spectra and maximum cross sections are very sensitive to either changes in the chromophore structure (mOrange vs mRFP) or mutations in chromophore surrounding (DsRed and mFruits). All red FPs show two pronounced 2PA transitions, the first peaking in the 1000 - 1100-nm region, and the second - near 700 - 760 nm. For each region we have found a mutant, which is 3 - 4 times two-photon brighter than the benchmark EGFP.
Searching for the two-photon brightest red fluorescent protein and its optimum excitation wavelength
Fluorescence In Vivo Imaging Based on Genetically Engineered Probes: From Living Cells to Whole Body Imaging IV, 2009
We study 2PA spectra of red fluorescent proteins (FPs), including DsRed2, mRFP, tagRFP, and mFruits in a wide range of excitation wavelengths, 600 -1200 nm. For evaluation of mature FP extinction coefficient and concentration we propose a pure optical method which is based on Strickler -Berg equation, relating fluorescence radiative lifetime with molecular extinction coefficient. 2PA spectra and maximum cross sections are very sensitive to either changes in the chromophore structure (mOrange vs mRFP) or mutations in chromophore surrounding (DsRed and mFruits). All red FPs show two pronounced 2PA transitions, the first peaking in the 1000 -1100-nm region, and the second -near 700 -760 nm. For each region we have found a mutant, which is 3 -4 times two-photon brighter than the benchmark EGFP.
Journal of Biomedical Optics, 2001
The imaging of living cells and tissues using laser-scanning microscopy is offering dramatic insights into the spatial and temporal controls of biological processes. The availability of genetically en- coded labels such as green fluorescent protein (GFP) offers unique opportunities by which to trace cell movements, cell signaling or gene expression dynamically in developing embryos. Two-photon laser scanning microscopy (TPLSM) is ideally suited to imaging cells in vivo due to its deeper tissue penetration and reduced phototoxicity; how- ever, in TPLSM the excitation and emission spectra of GFP and its color variants [e.g., CyanFP (CFP); yellowFP (YFP)] are insufficiently distinct to be uniquely imaged by conventional means. To surmount such difficulties, we have combined the technologies of TPLSM and imaging spectroscopy to unambiguously identify CFP, GFP, YFP, and redFP (RFP) as well as conventional dyes, and have tested the ap- proach in cell lines. In our approach, a liquid crystal tunable filter was used to collect the emission spectrum of each pixel within the TPLSM image. Based on the fluorescent emission spectra, supervised classifi- cation and linear unmixing analysis algorithms were used to identify the nature and relative amounts of the fluorescent proteins expressed in the cells. In a most extreme case, we have used the approach to separate GFP and fluorescein, separated by only 7 nm, and appear somewhat indistinguishable by conventional techniques. This ap- proach offers the needed ability to concurrently image multiple col- ored, spectrally overlapping marker proteins within living cells. Note: The technology and methods outlined in this paper were part of a patent licensed by Carl Zeiss and eventually sold as the META, an add-on that created a spectral confocal microscope. The invention and product won a 2002 Innovation award from R&D Magazine (the Oscars of Technology), as one of the best inventions of 2002. The META enabled users to do a lot of experiments not possible; every confocal microscope vendor now sells some device to provide similar perfomance.